Periodic Reporting for period 1 - EYEPOD (The vision-strike conversion: Neural control of the predatory strike behavior in stomatopods)
Periodo di rendicontazione: 2016-08-01 al 2018-07-31
Stomatopods utilize a super fast, power-amplified strikes for predation and defensive behaviours. The mechanics of the strike are such that potential energy from muscle contraction is stored in a spring that, when released, rotates the striking limb faster and with more power than would be possible from muscle action alone. The stomatopod strike is ballistic, which means it occurs so fast the movement cannot be modified or corrected once it is initiated. Thus, understanding the feed-forward neural controls stomatopods use to release their strike onto a target will reveal information for the development of ultra-fast movement technology and engineering.
While it is unknown which sensory cues are required to control strike systems, it is hypothesized that the complex visual system boasted by stomatopods may mediate strike control. The major problem addressed in this research is to identify descending neural controls for stomatopod strike release. A 2nd problem was to test which dimensions of visual information (if any) are used to control the strike release. Since stomatopods are not an established model for neurophysiological study, this research required several basic objectives in order to address the larger goals of this work: 1. Define the strike behaviour using high-speed video (hsv) recordings. 2. Identify the location of strike-control neurons via current injection. 3. Anatomically describe the neural circuitry involved in strike control and identify putative, large diameter axon targets for electrophysiological recording. 4. Develop an extracellular recording preparation for stomatopods.
While it is unknown which sensory cues are required to control strike systems, it is hypothesized that the complex visual system boasted by stomatopods may mediate strike control. The major problem addressed in this research is to identify descending neural controls for stomatopod strike release. A 2nd problem was to test which dimensions of visual information (if any) are used to control the strike release. Since stomatopods are not an established model for neurophysiological study, this research required several basic objectives in order to address the larger goals of this work: 1. Define the strike behaviour using high-speed video (hsv) recordings. 2. Identify the location of strike-control neurons via current injection. 3. Anatomically describe the neural circuitry involved in strike control and identify putative, large diameter axon targets for electrophysiological recording. 4. Develop an extracellular recording preparation for stomatopods.
Prior to the development of all experiments, l established access to my target species: Squilla mantis via local fisherman in Malaga, Spain. I also designed and built a field laboratory where I maintained animals, conducted electrophysiology experiments, and filled neurons for fluorescent microscopy in a local home. The majority of experiments with S. mantis were carried out during their available season in the fishing industry from November to March in both year 1 and year 2. During these field seasons, experiments were conducted in Spain (3-5 trips per year) and in Cambridge. The specific work achieved for each primary objective are as follows:
1.DEFINE STRIKE BEHAVIOR: Restrained animals were induced to strike via mechanical stimulation during HSV recordings. Strikes were induced both in air and in water to measure the effects of drag on the kinematics of the strike. Strike speeds were calculated using a custom code in Matlab and R. The results of this work suggests that mantis shrimp modulate the speed of their strikes (not all-or-nothing).
2.CURRENT INJECTION: different regions of the nervous system were stimulated to identify the location of strike control circuits. Strikes could only be induced by stimulating the circumesophogeal connectives (CEC). Lesion experiments demonstrate strikes may be induced independent of brain input. HSV analysis of current stimulated strikes demonstrate that only strike acceleration is engaged and not braking mechanism.
3. ANATOMY: Light microscopy of CECs revealed population of giant axons (>40um) in S. mantis. CEC and strike motor nerve axons were labelled with multiple fluorescent dyes to trace connections between the two nerves. Over 30 tissue samples were prepared for subsequent 3D imaged using 2 photon microscopy. CT scans generated high-resolution 3D scans of the striking appendages. These data served as a guide for muscular electrode placements and for a publication on the biomechanics the strike latch.
4. ELECTROPHYSIOLOGY: A stable, partially restrained extracellular recording preparation was designed Squilla mantis. The animal is restrained such that only the rotating appendage is free to move during a strike. Custom electrodes were used to record from the CEC. To monitor strike activity, EMG silver electrodes simultaneously record from the strike-driving muscles. Initial analyses identified descending neuronal units that respond to specific moving target visual stimuli. These same units also respond to mechanical stimulation of the antennae. While these data demonstrate presence of descending neurons with multi-sensory input, their targets remain unknown.
Figure 1. Diagram and unpublished data from S. mantis electrophysiological preparation. A Diagrams of experimental preparation viewed from top and the side (grey inset). (B) Light micrograph CEC crossection. * denote large diameter axons (>40um). D, dorsal; L, lateral. (C) Spontaneous spiking from CEC recording. D Spiking from same recording in response to moving target stimuli. Inset shows stimulus screen and region where most visual responses occurred (dashed ellipse, dorsal visual field). Green line, stimulus start; red line, stimulus stop. E Coactivity in CEC and lateral extensor muscle recordings during a spontaneous strike (not in response to stimuli). Strike movement occurs at approximately 2 s. Top, orange trace is CEC recording and bottom, red trace is EMG recording for C-E.
Figure 2. Angular velocity of strikes stimulated in a single animal via mechanical stimulation or current injection. A. Maximum angular velocity of mechanically stimulated strikes are greater than strikes forced by current stimulation to the CEC. B. Though the two stimuli produce different strike speeds, both current injection and mechanical stimulation accelerate the appendage at the same rate. Deceleration of the appendage is highly variable in current elicited strikes, suggesting that the braking mechanism of the strike is not recruited b
1.DEFINE STRIKE BEHAVIOR: Restrained animals were induced to strike via mechanical stimulation during HSV recordings. Strikes were induced both in air and in water to measure the effects of drag on the kinematics of the strike. Strike speeds were calculated using a custom code in Matlab and R. The results of this work suggests that mantis shrimp modulate the speed of their strikes (not all-or-nothing).
2.CURRENT INJECTION: different regions of the nervous system were stimulated to identify the location of strike control circuits. Strikes could only be induced by stimulating the circumesophogeal connectives (CEC). Lesion experiments demonstrate strikes may be induced independent of brain input. HSV analysis of current stimulated strikes demonstrate that only strike acceleration is engaged and not braking mechanism.
3. ANATOMY: Light microscopy of CECs revealed population of giant axons (>40um) in S. mantis. CEC and strike motor nerve axons were labelled with multiple fluorescent dyes to trace connections between the two nerves. Over 30 tissue samples were prepared for subsequent 3D imaged using 2 photon microscopy. CT scans generated high-resolution 3D scans of the striking appendages. These data served as a guide for muscular electrode placements and for a publication on the biomechanics the strike latch.
4. ELECTROPHYSIOLOGY: A stable, partially restrained extracellular recording preparation was designed Squilla mantis. The animal is restrained such that only the rotating appendage is free to move during a strike. Custom electrodes were used to record from the CEC. To monitor strike activity, EMG silver electrodes simultaneously record from the strike-driving muscles. Initial analyses identified descending neuronal units that respond to specific moving target visual stimuli. These same units also respond to mechanical stimulation of the antennae. While these data demonstrate presence of descending neurons with multi-sensory input, their targets remain unknown.
Figure 1. Diagram and unpublished data from S. mantis electrophysiological preparation. A Diagrams of experimental preparation viewed from top and the side (grey inset). (B) Light micrograph CEC crossection. * denote large diameter axons (>40um). D, dorsal; L, lateral. (C) Spontaneous spiking from CEC recording. D Spiking from same recording in response to moving target stimuli. Inset shows stimulus screen and region where most visual responses occurred (dashed ellipse, dorsal visual field). Green line, stimulus start; red line, stimulus stop. E Coactivity in CEC and lateral extensor muscle recordings during a spontaneous strike (not in response to stimuli). Strike movement occurs at approximately 2 s. Top, orange trace is CEC recording and bottom, red trace is EMG recording for C-E.
Figure 2. Angular velocity of strikes stimulated in a single animal via mechanical stimulation or current injection. A. Maximum angular velocity of mechanically stimulated strikes are greater than strikes forced by current stimulation to the CEC. B. Though the two stimuli produce different strike speeds, both current injection and mechanical stimulation accelerate the appendage at the same rate. Deceleration of the appendage is highly variable in current elicited strikes, suggesting that the braking mechanism of the strike is not recruited b
The exchange of expertise between myself, a stomatopod visual ecologist, and my mentor, Paloma Gonzalez-Bellido an established researcher studying the neuroethology of aerial insect predators, has resulted in a state-of-the art system for investigating the neurobiology of power-amplified movement. We are now equipped with new method to study extracellular electrophysiology, fluorescent neuron labelling and imaging, and biomechanics of the strike system in stomatopods. This research will continue in my new position at the University of Minnesota. At UMN I will pursue behavioral experiments in different species, building on the observations made during this fellowship. Without the work conducted on Squilla mantis during my fellowhip, the present research opportunity would not exist. The impact of this project on my career path is substantial. By developing my own independent research program, I am now qualified to apply for future faculty positions.
A tremendous amount of data was generated by this project, the analysis of which has subsequently delayed the progress of publication. I anticipate a minimum of three papers to result from this work soon after my transition to the University of Minnesota. This research is also directly linked to the development of new educational modules for low budget classrooms. During Summer 2018, I mentored an undergraduate intern at the company Backyard Brains (AnnArbor, MI, USA) who is developed a system for recording from freely moving stomatopod crustaceans. This project will lead to the development of an out-of-the-box kit for students to study limb movement in the classroom. The MSC fellowship directly led to the development of this new educational tool that integrates concepts of neurobiology, physiology, and biomechanics.
A tremendous amount of data was generated by this project, the analysis of which has subsequently delayed the progress of publication. I anticipate a minimum of three papers to result from this work soon after my transition to the University of Minnesota. This research is also directly linked to the development of new educational modules for low budget classrooms. During Summer 2018, I mentored an undergraduate intern at the company Backyard Brains (AnnArbor, MI, USA) who is developed a system for recording from freely moving stomatopod crustaceans. This project will lead to the development of an out-of-the-box kit for students to study limb movement in the classroom. The MSC fellowship directly led to the development of this new educational tool that integrates concepts of neurobiology, physiology, and biomechanics.